http://hdl.handle.net/1957/19990 Fri, 24 May 2013 14:46:26 GMT 2013-05-24T14:46:26Z http://hdl.handle.net/1957/38287 Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C‐MYC depletion Mannava, Sudha; Moparthy, Kalyana C.; Wheeler, Linda J.; Leonova, Katerina I.; Wawrzyniak, Joseph A.; Bianchi-Smiraglia, Anna; Berman, Albert E.; Flanagan, Sheryl; Shewach, Donna S.; Zeitouni, Nathalie C.; Gudkov, Andrei V.; Mathews, Christopher K.; Nikiforov, Mikhail A. The down‐regulation of dominant oncogenes, including C‐MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC‐depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC‐depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribonucleosides to culture media substantially inhibited DNA damage and senescence‐associated phenotypes caused by C‐MYC depletion. Our data demonstrate the essential role of TS and RR in C‐MYC‐dependent suppression of senescence in melanoma cells. This is the publisher’s final pdf. The published article is copyrighted by Impact Journals LLC. and can be found at: http://www.impactaging.com/index.html. Sat, 01 Dec 2012 00:00:00 GMT http://hdl.handle.net/1957/38287 2012-12-01T00:00:00Z http://hdl.handle.net/1957/37950 Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression Saldivar, Joshua C.; Miuma, Satoshi; Bene, Jessica; Hosseini, Seyed Ali; Shibata, Hidetaka; Sun, Jin; Wheeler, Linda J.; Mathews, Christopher K.; Huebner, Kay Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability. This is the publisher’s final pdf. The published article is copyrighted by Public Library of Science and can be found at: http://www.plos.org/. Thu, 29 Nov 2012 00:00:00 GMT http://hdl.handle.net/1957/37950 2012-11-29T00:00:00Z http://hdl.handle.net/1957/37897 Regulation of antimicrobial peptide gene expression by nutrients and byproducts of microbial metabolism Campbell, Yan; Fantacone, Mary L.; Gombart, Adrian F. Background: Antimicrobial peptides (AMPs) are synthesized and secreted by immune and epithelial cells that are constantly exposed to environmental microbes. AMPs are essential for barrier defense and deficiencies lead to increased susceptibility to infection. In addition to their ability to disrupt the integrity of bacterial, viral and fungal membranes, AMPs bind lipopolysaccharides, act as chemoattractants for immune cells and bind to cellular receptors and modulate the expression of cytokines and chemokines. These additional biological activities may explain the role of AMPs in inflammatory diseases and cancer. Modulating the endogenous expression of AMPs offers potential therapeutic treatments for infection and disease. Methods: The present review examines published data from both in vitro and in vivo studies reporting effects of nutrients and byproducts of microbial metabolism on the expression of antimicrobial peptide genes in order to highlight an emerging appreciation for the role of dietary compounds in modulating the innate immune response. Results: Vitamins A and D, dietary histone deacetylases and byproducts of intestinal microbial metabolism (butyrate and secondary bile acids) have been found to regulate the expression of AMPs in humans. Vitamin D deficiency correlates with increased susceptibility to infection and supplementation studies indicate an improvement in defense against infection. Animal and human clinical studies with butyrate indicate that increasing expression of AMPs in the colon protects against infection. Conclusion: These findings suggest that diet and/or consumption of nutritional supplements may be used to improve and/or modulate immune function. In addition, byproducts ofgut microbe metabolism could be important for communicating with intestinal epithelial and immune cells, thus affecting the expression of AMPs. This interaction may help establish a mucosal barrier to prevent invasion of the intestinal epithelium by either mutualistic or pathogenic microorganisms. This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Springer and can be found at: http://link.springer.com/journal/394. Sat, 01 Dec 2012 00:00:00 GMT http://hdl.handle.net/1957/37897 2012-12-01T00:00:00Z http://hdl.handle.net/1957/37666 The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle Winzeler, Elizabeth A. The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle were investigated by studying the time-resolved fluorescence anisotropy decays of ethidium bromide intercalated into the DNA of the core particle. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. The anisotropy decays were collected using the method of time-resolved single-photon counting and were fit to a model developed by J. M. Schurr (Schurr, 1984) using a non-linear least squares fitting algorithm developed by the author for this purpose. It was shown that below a binding ratio of 0.1 there was no demonstrable change in the anisotropy as a function of binding ratio. Our results show, that the apparent torsional flexibility of DNA of to a nucleosome core particle is dependent on the number of base pairs of the DNA between points of attachment to the histone core. If this number is as high as 30 base pairs, then the torsional flexibility of DNA on a nucleosome core particle is as high or higher than DNA free in solution. Also, for reasonable values of N, the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the DNA on a core particle is highly constrained in its motions. The hydrogen ion concentration was shown to have a substantial effect on the fluorescent anisotropy decays, particularly in the early regions of the decay. These analyses indicated that the observed change could be attributed to either a loosening of the contacts between the DNA and the histone core, or a relaxing of the torsional flexibility of the DNA. Graduation date: 1991 Fri, 20 Jul 1990 00:00:00 GMT http://hdl.handle.net/1957/37666 1990-07-20T00:00:00Z